1. A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a
- Author
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Florian Katzmeier, Lukas Aufinger, Friedrich C. Simmel, Matthias Lenz, Benedikt Dürr, Michael Heymann, Igor Styazhkin, Ludwig Bauer, Sven Klumpe, Maximilian N. Honemann, Jorge Quinteiro, Dawafuti Sherpa, and Aurore Dupin
- Subjects
Luminescence ,Transcription, Genetic ,Light ,Hydrolases ,Computer science ,CRISPR-Associated Proteins ,Biosensing Techniques ,Biochemistry ,01 natural sciences ,Signal ,chemistry.chemical_compound ,Filter Paper ,Limit of Detection ,Nucleic Acids ,Fluorescein ,0303 health sciences ,Physics ,Electromagnetic Radiation ,Detector ,Fluorescence ,Enzymes ,Laboratory Equipment ,Signal Filtering ,RNA, Bacterial ,Bioassays and Physiological Analysis ,Artificial Light ,visual_art ,Physical Sciences ,visual_art.visual_art_medium ,Engineering and Technology ,Medicine ,Computer hardware ,Research Article ,Nucleic acid detection ,Nucleases ,Science ,Green Fluorescent Proteins ,Equipment ,In Vitro Techniques ,Research and Analysis Methods ,Fluorescence spectroscopy ,03 medical and health sciences ,Ribonucleases ,Bacterial Proteins ,DNA-binding proteins ,Instrumentation (computer programming) ,Colorimetric Assays ,030304 developmental biology ,Detection limit ,Biology and life sciences ,business.industry ,010401 analytical chemistry ,Proteins ,In vitro ,0104 chemical sciences ,chemistry ,Filter (video) ,Signal Processing ,Electronic component ,Enzymology ,Nucleic acid ,CRISPR-Cas Systems ,Biochemical Analysis ,business - Abstract
Point-of-care testing (POCT) in low-resource settings requires tools that can operate independent of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably detected less than 10 nM fluorescein concentrations (with a lower limit of detection of ≈6.8 nM), which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs in the nanomolar range using a Cas13a-based fluorescence assay. Cas13a is a RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device architecture could be a valuable educational tool that integrates hardware, software and biochemistry concepts.
- Published
- 2019
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